Globulin determination

ABSTRACT

A STABLE REAGENT FOR THE DETERMINATION OF GLOBULINS IN BIOLOGICAL FLUIDS IS PREPARED BY DISSOLVING PARA-TOLUENESULTONIC ACID AND A CUPRIC SALT SUCH AS CUPRIC SULFATE PENTAHYDRATE IN ACETIC ACID, OR PREFERABLY, IN A MIXTURE OF ACETIC ACID AND SULFURIC ACID. THE REAGENT HAS IMPROVED STABILITY, SENSITIVITY AND SPECIFICITY PROPERTIES AND CAN BE EMPLOYED IN A ONE-TUBE PROCEDURE WITH THE BIURET COLOR REACTION FOR RAPID DETERMINATION OF TOTAL PROTEIN AS WELL AS GLOBULIN. METHODS OF PREPARING THE REAGENT AND USING I IN THE DETERMINATION OF GLOBULINS ARE DISCLOSED.

UnitedStates Patent 3,627,468 GLOBULIN DETERMINATION Harry Goldenberg, Santa Monica, and Donald R. Wybenga, Alhambra, Calif., assignors to The Dow Chemical Company, Midland, Mich. No Drawing. Filed June 30, 1969, Ser. No. 837,896 Int. Cl. G01n 31/16, 31/22, 21/24 US. Cl. 23-- 230 B 11 Claims ABSTRACT OF THE DISCLOSURE A stable reagent for the determination of globulins in biological fluids is prepared by dissolving para-toluenesultonic acid and a cupric salt such as cupric sulfate pentahydrate in acetic acid, or preferably, in a mixture of acetic acid and sulfuric acid. The reagent has improved stability, sensitivity and specificity properties and can be employed in a one-tube procedure with the biuret color reaction for rapid determination of total protein as well as globulin. Methods of preparing the reagent and using it in the determination of globulins are disclosed.

FIELD OF THE INVENTION The invention is directed to a single reagent for the determination of globulins in biological fluids such as serum, plasma, cerebrospinal fluid or the like and to a method for determining the ratio of globulin to total protein in the fluid.

DESCRIPTION OF THE PRIOR ART The biuret reaction has long been employed as an accepted method for the determination of the total protein in biological fluids. In this procedure, the proteins in the biological fluid react with cupric ions in alkaline solu tion to form a purple colored product. The intensity of the purple color is proportional to the concentration of protein in the biological fluid. Measurement of the intensity of the color is typically carried out by measuring the optical density, absorbance or transmittance of the purple color with light having a wavelength between 450 and 650 millimicrons. Conventional instruments such as spectrophotometers or colorimeters. are typically employed in such color measurements of the biuret reaction mixture. In a typical procedure, the intensity of color formed by the reaction of the protein and the biuret reagent can be compared against a reagent blank, comprising the biuret reagent in the absence of protein, and distilled water. The biuret reaction is described further in Henry, Clinical Chemistry: Principles and Techniques, page 182., Hoeber Division of Harper & Rowe, New York (second printing, 1964) and Henry et al., Analytical Chemistry, 29, 1491-1495 (1957).

In a variety of situations, it is desirable to determine not only the total protein concentration, but the concentration of particular types of protein such as globulins or albumins. The biuret reaction involving the same type of alkaline cupric ion reagent has often been employed again in determination of specific protein fractions after fractionation of the protein. The fractionation is carried out as a separate step by any technique capable of giving the desired separation of globulins from the remainder of the protein. Electrophoretic separation techniques such as starch gel, agar gel or paper electrophoresis provide excellent separation of fractions; however, such procedures require extensive and costly apparatus and considerable time and skill is required for the separation.

Salt fractionation has been employed to determine globulins in serum by the biuret reaction. In this technique, the globulins are precipitated with sodium sulfite leaving the albumins in solution. The albumin concentration can be determined by the biuret reaction and the globulin concentration determined by difference. Alternately, the precipitated globulins can be redissolved separately and determined by the biuret reaction. The salt fractionation technique of Wolfson, Cohn et al., American Journal of Clinical Pathology, Technical Section, 18, 723 (1948) is an accepted procedure of this type and gives results comparable to those involving electrophoresis. Henry, op cit. supra, pages 207-211.

None of the prior art procedures has been entirely satisfactory. It would be desirable to provide a reagent and method which would permit the determination of both total protein and globulin in a single sample without requiring fractionation, electrophoresis or the like. It would also be desirable to provide a reagent and method for the determination of globulin in biological fluids which is sufficiently sensitive and specific to permit direct determinations on unmodified fluids without interference by colors due to other substances such as bilirubin. It would also be desirable to provide a globulin reagent and method which can be employed in a mixture of biological fluid and biuret reagent so that protein and globulin determinations can be made in a simple direct two-step operation using a single tube and a single sample, while providing results comparable to those obtained when electrophoresis or salt fractionation steps are included.

SUMMARY OF THE INVENTION This invention is directed to a novel single reagent for the determination of globulins in biological fluids and to a method for using the same.

It is an object of this invention to provide a single reagent for the determination of globulin in biological fluids such as serum or plasma or the like which can be prepared well in advance of its use and which remains stable for long periods of time. Another object of this invention is to provide a reagent and method which produces a specific measurable color from globulins so that the color measurement can be made without interference by color due to the presence of other substances such as bilirubin and the like. Another object is to provide a reagent and method which will permit rapid simple determinations of both total protein and globulin concentration to be carried out in the same tube. A further object is to povide a reagent and method which produces a measurable color with globulin in the presence of other proteins so that the quantitative results obtained are comparable to those obtained by prior art methods involving electrophoresis or salt fractionation of the biological fluid sample. Other objects and advantages of the present invention will be apparent on consideration of the following description and claims.

The reagent for use in the determination of globulins in accordance with the invention comprises a mixture of para-toluenesulfonic acid, acetic acid and cupric ion, and optionally, an analytically-acceptable strong acid. More particularly, the reagent comprises a colorimetric amount of para-toluenesulfonic acid and a sensitizing amount (color-enhancing amount) of cupric ion dispersed in acetic acid. The reagent can also include a decolorizing amount of an analytically-acceptable strong acid.

The term "analytically-acctptable strong acid is employed herein to mean those organic and inorganic acids having an ionization constant greater than about 0.001, which are compatible with the other ingredients of the reagent composition, which are not detrimentally reactive with proteins, or with globulins in particular, and which do not interfere with the color reaction. The acid employed can be a strong mineral acid such as sulfuric acid, sulfamic acid or polyphosphoric acid or mixtures thereof. The preferred analytically-acceptable strong acid is sulfuric acid.

The cupric ion can be supplied in any convenient form to provide for solution thereof in the composition. Generally, a soluble salt of copper such as cupric sulfate, cupric sufate pentahydrate, cupric acetate or the like is convenient, and cupric sulfate is particularly preferred.

The novel reagent composition forms a colored product when mixed with globulins. The ingredients of the reagent composition cooperate to disperse globulins contained in a sample throughout the reagent composition and to facilitate the formation of the colored product. Thus, the reagent composition can be employed to determine the presence or absence of globulins in biological fluids. The intensity of the color formed by admixture of the reagent composition of the invention with globulins or biological fluid containing the same is proportional to the amount of globulins present. Thus, the color can be measured to give an, accurate rapid quantitative measurement of globulin concentration. In addition, the reagent is specific for globulins and extremely sensitive, permitting quantitative analyses for globbulins to be performed directly on biological fluids containing other proteins Without substantial interference by colors due to the present of other proteins or substances such as bilirubin. Moreover, the novel reagent composition is stable in storage for substantial periods of time. Use of the color reagent to determine concentrations of globulins gives accurate results which are comparable to those obtained with methods requiring substantially more time for analysis and requiring additional analytical separation procedures such as electrophoresis or salt fractionation of the sample. The novel globulin reagent can also be employed in a novel process wherein the concentration of globulins is determined in the same tube as a biuret protein determination, without separation of proteins by salt fractionation or the like. The novel reagent and method can be employed to determine total protein concentration and globulin concentration in successive operations with the identical sample, even when the intensity of color is measured for light of the same wavelength in both determinations.

In preparing the reagent composition of the invention, the para-toluenesulfonic acid and a copper salt suitable for supplying the cupric ion are dispersed in acetic aicd in any order or fashion. When an analytically-acceptable strong acid is employed, it is then mixed with the resulting composition. In the preferred procedure, all the acids as Well as the copper salt are reagent grade materials.

In a convenient procedure, para-toluenesulfonic acid is mixed with glacial acetic acid at temperatures from about 20 to about 30 C. A solution of the copper salt in a small amount of water is added to the acid mixture. Sulfuric acid is thereafter mixed with the organic acid solution of the copper salt. In the preferred procedure, the sulfuric acid is added portionwise with stirring or agitation and the mixture is cooled in an ice bath during and after each addition. The reagent thus prepared is stable for periods of several months when stored under refrigeration.

The presence of a colorimetric amount of paratoluenesulfonic acid in the reagent composition is critical and essential to the use of the composition in globulin determination. Suflicient para-toluenesulfonic acid must be present in the ultimate reagent composition so that a measurable color is produced when the reagent composition is mixed with globulins or a biological fluid containing the same. For quantitative determinations, it is essential that the para toluenesulfonic acid be employed not only in an amount suflicient to provide a detectable color, but also in an amount which is sufiicient so that a colored product is formed with all the globulins present, thus providing an intensity of color proportional to the amount of globulin. The ultimate mixture for quantitative determinations thus comprises acetic acid, the cupric ion, a minor amount of globulin and an amount of para toluenesulfonic acid sufficient to provide an intensity of color substantially proportional to the amount of globulin. In the present specification and claims, the phrase colorimetric amount is employed to designate that concentration of para-toluenesulfonic acid in the composition which provides a measurable color with globulin, the intensity of said color being substantially proportional to the amount of globulin. When insufficient par-a-toluenesulfonic acid is employed, qualitative results can be obtained, but the quantity of globulins in excess over that required to form the colored product is not measured. In particular applications, whether or not a colorimetric amount of para-toluenesulfonic acid has been employed can be determined by the simple expedient of measuring the color produced with varying amounts of globulin. The reagent and sample are preferably mixed to provide an excess of para-toluenesulfonic acid over that required to form the colored product to ensure that the intensity of color is limited only by globulin concentration. The use of such an excess of para-toluenesulfonic acid ensures that the intensity of color is proportional to the amount of globulin.

The presence of a sensitizing amount of cupric ion in the reagent composition is also critical and essential in the use of the reagent in direct determinations of globulin. Suflicient of the cupric ion must be present in the ultimate reagent composition so that the color produced in the reaction of globulin with the other ingredients is detectably increased in intensity. The increased intensity in color permits the use of the reagent in quantitative determinations employing very small amounts such as 0.05 to 1.0 milligram of globulin, even in the presence of other proteins, bilirubin and biuret reagents or the like. Both the colored reaction product and the cupric ion absorb light of similar Wavelengths. Thus, concentration of cupric ion must not be so great as to obscure the color formed With globulin. In the present specification and claims, the term sensitizing amount of cupric ion is employed to designate that concentration of cupric ion which detectably enhances the color produced in the reaction with globulins and thus significantly increases the sensitivity of the para-toluenesulfonic acid to small amounts of globulin without creating sufiicient cupric ion color to obscure the desired globulin reagent color. In particular applications, the desirable sensitizing amount of cupric ion to be employed can be ascertained by comparing the intensity of color obtained when reagent compositions containing varying amounts of cupric ion are mixed with small amounts of globulin, such as from 0.01 to 0.1 or more milligrams of globulin per milliliter of ultimate mixture. The amount of cupric ion should be in- SLllfiClCIlt to give the reagent alone a depth of color due to cupric ion equivalent to an absorbance of more than about 0.2 for light of a Wavelength from 480 to 600 millimicrons. When the reagent alone, Without the addition of globulin, has an absorbance substantially greater than about 0.2, the cupric ion color can obscure the globulin reagent color, making the reagent less sensitive to loW globulin concentrations.

The acetic acid to be employed in preparing the reagent composition is glacial acetic acid of a high degree of purity. Glacial acetic acid generally contains trace amounts of glyoxylic acid, which may be present therein as a product of oxidation of the acetic acid. The trace 7 amounts of glyoxylic acid present in the acetic acid are less than about parts by volume of glyoxylic acid per million parts by volume of acetic acid and are generally too slight to be accurately measured by quantitative procedures. The presence of such trace amounts of glyoxylic acid in the acetic acid has no detrimental effect on the reagent and method of the invention. Since trace amounts of glyoxylic acid are generally present in acetic acid, the term acetic acid, as employed herein, is inclusive of acetic acid containing a trace amount of glyoxylic acid.

The stability of. the reagent composition is greatly enhanced by the addition thereto of a decolorizing amount of an analytically-acceptable strong acid, and the presence of a decolorizing amount of such acid is essential for the use of the reagent together with the 'biuret reaction in a one-tube analytical process. The term decolorizing amount as employed herein refers to that amount of analytically-acceptable strong acid which enables the reagent of the invention to produce a measurable color on admixture of the reagent with a mixture of globulin or biological fluid containing the same and biuret reagent, with the depth of the color being proportional to the concentration of globulins in the mixture. The analytically-acceptable strong acid removes the biuret reaction color when the reagent composition is mixed with a biological fluid containing a biuret reagent, thus decolorizing the mixture and minimizing interference between the biuret color and the globulin reagent color. When insufficient analytically-acceptable acid is employed, the resulting reagent provides excellent quantitative determinations of globulin when mixed with biological fluids directly. However, when the biological fluid has been previously mixed with biuret reagent, as in a prior determination of total protein, quantitative determinations of globulin cannot be performed without additional treatment steps.

Whether or not a decolorizing amount of the strong acid is employed in particular situations involving a certain biuret reaction mixture can be determined by employing the reagent with samples containing known amounts of globulin and checking to determine if the color intensity is substantially proportional to globulin concentration. The use of a decolorizin g amount of the analytically-acceptable acid also enhances stability of the composition, and reagent compositions containing a decolorizing amount of an analytically-acceptable strong acid are much more stable in storage and less sensitive to temperature changes in storage than the reagent composition which contains no analytically-acceptable acid.

In general, good results are obtained with reagent compositions containing a colorimetric amount of paratoluenesulfonic acid at concentrations of from about to about 20 percent (weight in grams of para-toluenesulfonic acid by volume in milliliters of the ultimate reagent mixture) and with a sensitizing amount of cupric ion equivalent to from about 0.04 percent by weight of cupric sulfate pentahydrate by volume of ultimate reagent to suflicient cupric ion to provide an optical density at 480600 millimicrons of about 0.2 in the ultimate mixture. Generally, cupric ion concentrations equivalent to from about 0.04 to about 0.2 percent by weight of cupric sulfate pentahydrate by volume of ultimate mixture are preferred.

The liquid acid ingredients can be present in the reagent in approximately the percentages given below:

Acetic acid from about 100 to about 70 percent by volume; and

Analytically-acceptable strong acid from zero to about 30 percent by volume.

When quantitative determinations of total protein as well as globulins are desired, the liquid acid ingredients are preferably present as a mixture containing from about 5 to about 25 percent by volume of analytically-acceptable strong acid in the acetic acid.

In a preferred reagent composition, the ingredients are employed in the following proportions:

Sulfuric acid from about to about percent by volume;

Acetic acid from about 80 to about 90 percent by volume;

Para-toluenesulfonic acid from about 5 to about 15 percent (weight in grams by volume in milliliters of acetic and sulfuric acids); and

Cupric sulfate pentahydrate from about 0.04 to about 0.15 percent (Weight in grams by volume in milliliters of acetic acid and sulfuric acid) employed.

The preferred reagent composition can additionally include a small amount of water, generally from about 0.1

to about one percent by volume, employed when the cupric salt is added in aqueous solution.

In the qualitative or quantitative determination of globulin with the reagent of the invention, the reagent is mixed with a sample, generally a biological fluid or a globulin-containing substance such as a standard solution. In quantitative operations, the reagent is mixed with a minor amount of a biological fluid containing globulin. The biological fluid can be an extract, as from a tissue homogenate or the like, or it can be an animal body fluid such as blood, plasma, serum, lymphatic fluid, bile, cerebrospinal fluids or the like. The biological fluid can be employed directly as a sample or it can be treated by conventional procedures such as dilution, concentration, filtration, centrifugation, extraction or the like.

In a particularly useful embodiment, the preferred reagent of the invention containing the analyticallyacceptable strong acid ingredient, is mixed with a biuret reaction mixture comprising a biological fluid and sufficient of a biuret reagent to provide a color proportional to the concentration of protein therein. The biuret reagent can be any of the cubic Sulfate reagents known to be suitable for determination of protein in biological fluids, such as the reagent of Henry et al., Anal. Chem., 29, 1491 (1957). A convenient biuret reagent comprises an aqueous solution of about 0.3 to 0.4 percent (weight by volume) of cupric sulfate pentahydrate, about 3 to 4 percent (weight by volume) of sodium citrate and 1.5 to 2.5 percent (weight by volume) of sodium carbonate. The biuret reaction mixture is prepared by conventional procedures such as that of Henry et a1. Typically, the biuret reagent is mixed with a small amount of a base such as one part by volume of biuret reagent to from 0.1 to 0.2 part by volume of 2.5 Normal sodium hydroxide. The basic mixture is then mixed with a biological fluid conveniently in proportions of about one part by volume of biological fluid to about 20 to 50 parts by volume of basic biuret mixture. The mixture can be heated to a temperature of about 5070 C. for a few minutes, cooled and held while the biuret reaction color is measured in a spectro photometer or colorimeter typically employing light having a wavelength of 480 to 600 millimicrons. The mixture can be held for additional periods before it is mixed with the globulin reagent. Alternately, it can be mixed with the globulin reagent immediately after the biological fluid and biuret reagent are mixed.

In determining globulin, the reagent is mixed thoroughly with a minor proportion of the biological fluid sample, conveniently in the proportions of about one part by volume of biological fluid to about 40 to about 300 parts by volume of globulin reagent. When the sample is a mixture of biological fluid and biuret reagent, from about three to about fifteen parts by volume of the globulin reagent composition are admixed with each part by volume of the sample mixture. The reagent and sample can be mixed in receptacles of a particular predetermined optical density or absorbance such as the tubes and cuvets conventionally employed with colorimeters or spectrophotometers, if desired. When the sample is a mixture of biological fluid and biuret reagent prepared in such a tube for determination of the total protein concentration, the same tube can be employed. In such a case, depending on the volume of the sample mixture and the tube, it may be desirable to discard a measured portion of the biuret reaction mixture from the tube before adding the desired amount of globulin reagent composition in order to avoid spillage or overfilling of the tubes.

The reagent composition and globulin combine at elevated temperatures (about 50l10 C.) to form a colored product with a depth of color proportional to the amount of globulin. The exact temperature and time for heating are not critical when only qualitative determinations are desired. However, the quantitative accuracy of the method of the invention is greatly enhanced by controlling the time and temperature of heating. For rapid quantitative procedure, the mixture is heated to a temperature between about 90 and about 110 C. in from about to 15 minutes after heating is begun, and the mixture can then be cooled to a temperature below the above-stated temperature range within about three to about five minutes after heating has ceased.

The heating step can be conveniently carried out by placing tubes containing the mixture of reagent and sample in a conventional tube heating block at a temperature of 100 C. for from 5 to 15 minutes. In a convenient procedure for cooling the mixture, the tubes are removed from the heating block and immersed in a cold water bath for from three to five minutes. Other conventional means for heating and cooling the tubes can be employed such as hot water or oil baths, ice baths and the like.

The color of the mixture can be measured by any means which will give an accurate measurement of the intensity of color. Preferably, a spectrophotometer or a colorimeter is employed. When the method is carried out in a colorimeter or spectrophotometer tube or cuvet, the mixture can then be placed in the instrument and its absorbance or transmittance determined. Otherwise, an aliquot portion of the mixture can be placed in such a tube and the intensity of color is determined in a colorimeter or spectrophotometer. In such operations, it is preferred to determine the absorbance or percent transmittance of the sample with light having a Wavelength between about 480 millimicrons and 600 millimicrons. When the sample is a mixture of biological fluid and biuret reagent prepared for determination of total protein, it is convenient to employ the same colorimeter or spectrophotometer and light of the same wavelength employed in any prior total protein determination. The amount of globulin present in the sample can then be determined by a comparison of the percent transmittance or absorbance observed for the sample with the measurements obtained when samples containing known amounts of globulin are employed, or with conversion charts or tables prepared from such data. I

In making the colorimeter or spectrophotometer determinations, it is desirable to employ the readings obtained on the color reagent alone and in the absence of any globulin and to thus measure the difference in absorbance (optical density) or percent transmittance between the sample and the pure reagent (reagent blank).

It is also desirable to employ a globulin standard when employing the reagent of the invention to determine the amount of globulin present in a sample of biological fluid. In this procedure, a standard sample is prepared to contain a known amount of globulins, and this sample is mixed with the reagent in the same predetermined proportions and treated in the identical procedure as the sample of biological fluid containing an unknown amount of globulins. For best results, the globulins employed should originate from the same species as the biological fluid. Thus, for analysis of bovine fluids, a bovine globulin standard should be used or for human fluids, a human globulin standard should be used and so forth. The simultaneous use of a globulin standard substantially eliminates the effect of procedural deviations. The employment of the standard also permits the calculation of globulin concentration in the sample of biological fluid by comparison of the readings obtained with the standard and the sample. When the globulin determination is to be made in the same tube as a total protein determination, it is convenient to use a single tube containing both globulin and other protein to serve as a protein standard for the biuret reaction and also as a globulin standard.

In a convenient procedure, each sample or group of samples to be analyzed, a globulin standard or protein and globulin standard and a reagent blank composition comprising the reagent alone are treated simultaneously. Additional biological fluid sample tubes can be prepared so long as all tubes can be heated and cooled at the same temperatures simultaneously. The intensity of color for the globulin standard and the sample tubes is then measured, taking account of the reagent blank, and the concentration of globulins in the sample is obtained by com.- paring the intensity of color in the sample tube with the intensity of color produced by the known concentration of globulin in the standard.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The following examples illustrate the present invention but are not to be construed as limiting the same.

EXAMPLE 1 20 grams of cupric sulfate pentahydrate are dissolved in milliliters of water and the solution is diluted with water to a total volume of milliliters and mixed well. Five milliliters of the resulting solution are then mixed with a solution of grams of paratoluenesulfonic acid in 900 milliliters of glacial acetic acid, and the mixture is stirred and diluted with glacial acetic acid to a total volume of 1000 rnillilters. The resulting reagent is stirred and mixed well and employed in the determination of globulin.

Other globulin reagent compositions can be similarly prepared in accordance with the procedure described above by employing cupric acetate or anhydrous cupric sulfate in lieu of the cupric sulfate pentahydrate. The

compositions can be mixed directly with biological fluids in the determination of globulin, or they can be mixed with sulfuric acid before use, or they can be employed in determining globulin concentration in a mixture of biological fluid and biuret reagent.

For example, in substantially the same procedure as described above, a reagent composition is prepared by dissolving 100 grams of para-toluenesulfonic acid in 700 milliliters of glacial acetic acid, mixing the solution with 2 grams of cupric sulfate pentahydrate and mixing the resulting solution with 300 milliliters of sulfuric acid.

In substantially the same procedure, a reagent composition is prepared by dissolving grams of paratoluenesulfonic acid in 875 milliliters of glacial acetic acid, adding six milliliters of a 20 percent (weight by volume) aqueous solution of cupric acetate monohydrate, chilling the resulting solution and mixing the solution thoroughly with 125 milliliters of sulfuric acid.

In substantially the same procedure, 60 grams of paratoluenesulfonic acid are dissolved in 600 milliliters of glacial acetic acid. Twenty milliliters of an aqueous solution containing 20 percent (weight by volume) of cupric sulfate are added to the mixture and dispersed therein. The mixture is then diluted to a total volume of 950 milliliters with glacial acetic acid. The resulting solution is chilled in an ice bath and 50 milliliters of polyphosphoric acid are mixed therewith to prepare a reagent composition.

EXAMPLE 2 5.0 milliliters of the first above-described reagent of Example 1 are placed in each of three photometer tubes, A, B and C. 50 microliters of human serum containing an unknown amount of globulin and total protein are added to tube A. 0.050 milliliter of a solution containing 7.0 grams of protein, 3 grams of which are human globulins, in 100 milliliters of normal saline solution is added to tube B to provide a globulin standard. No serum or protein solution is added to tube C and tube C is employed as a reagent blank. The mixtures in tubes A and B are mixed thoroughly by shaking twenty times (about 10 seconds). All three tubes are then simultaneously placed in a heating block set at 100 C. and heated for ten minutes, at which time the temperature in each tube is approximately 95 100 C. The tubes are then removed simultaneously and immersed in a cold water bath for five minutes, during which time the temperature of the contents of each tube is lowered to about 25 C. The tubes are removed from the water, dried and the contents of each tube are mixed by inverting the tube several times. Tube C is placed in a colorimeter with a filter for light having a Wavelength of 540 millimicrons. The indicating device on the colorimeter has been previously set at zero absorbance for a similar tube containing distilled water. The absorbances of tubes A, B and C are then read on the colorimeter and recorded. The concentration of globulin in the sample in grams of globulin per 100 milliliters of sample is calculated by multiplying the difference between the absorbance of the sample (A) and that of the reagent blank (C) by 3 and dividing the product by the difference between the absorbance of the globulin standard (B) and that of the reagent blank (C).

The same procedure described above is carried out with 50 microliters of human plasma as the sample rather than with serum. The difference between result obtained for plasma less the globulin concentration of the serum is recorded as fibrinogen concentration.

The results obtained are in excellent agreement with the results obtained by determination of globulin on identical serum and plasma samples by the salt fractionation method of Wolfson, Cohn et a1.

EXAMPLE 3 1.24 kilograms of para-toluenesulfonic acid are dissolved in 9 liters of glacial acetic acid. 50 milliliters of a 20 percent (weight by volume) solution of cupric sulfate pentahydrate in water is mixed with the paratoluenesulfonic acid solution and the mixture is diluted with additional glacial acetic acid to a total volume of 10 liters. 8.5 liters of the resulting solution are mixed well with 1.5 liters of concentrated sulfuric acid to prepare a globulin reagent.

EXAMPLE 4 S'xty samples of human serum are collected and each portion is divided into two identical portions to provide two separate series, I and II, of serum samples, and each of which contains an unknown concentration of protein, and an unknown ratio of albumin to globulin. The serum samples of series I are analyzed for total protein concentration and globulin concentration by the following procedure:

Three milliliters of a biuret reagent, comprising 0.35 percent cupric sulfate pentahydrate, 3.5 percent sodium citrate and 2.0 percent sodium carbonate (weight by volume of aqueous solution), are placed in each of three tubes, A, B and C. 0.5 milliliter of aqueous 2.5 normal sodium hydroxide solution is added to each tube and the solutions are mixed by capping and inverting the tubes. 0.10 milliliter of a sample of serum from series I is added to tube A. A standard solution containing 7.5 grams of total protein per 100 milliliters (4.5 grams of human albumin and 3.0 grams of human globulin per 100 milliliters) is prepared. 0.1 milliliter of this standard solution is added to tube B to provide a protein and globulin standard. No serum or protein solution is added to tube C and tube C is employed as a reagent blank. The mixtures in tubes A and B are mixed thoroughly by shaking for at least five seconds. All three tubes are then simultaneously placed in a heating block set at 100 C. and heated for 1.0 minute at which time the temperature in each tube is approximately 60 C. The tubes are then removed simultaneously and immersed in water having a temperature not higher than C. for three minutes, during which time the temperature of the contents of each tube is lowered to about C. The tubes are then removed from the water, dried and the contents of each tube are mixed by inverting the tube twice. The absorbances of tubes A, B and C are read on a colorimeter with a filter for light having a wavelength of 540 millimicrons. The indicating device on the colorimeter has been previously set to read zero absorbance for distilled water. The absorbances of tubes A, B and C are read on the colorimeter and recorded. The concentration of total protein in the sample in grams of protein per 100 milliliters of sample is calculated by multiplying the difference between the absorbance of the sample (A) and that of the reagent blank (C) by 7.5 and dividing the product by the difference between the absorbance of the standard (B) and that of the reagent blank (C). After the absorbances of tubes A, B and C are read and recorded. 2.5 milliliters of the contents are removed from each tube and discarded. 5 milliliters of globulin reagent of Example 3 is then added to each of tubes A, B and C and mixed with the contents remaining thereinby inverting the tubes a few times.

All three tubes are then simultaneously placed in a heating block set at 100 C. and heated for ten minutes, at which time the temperature in each tube is approximately -100 C. The tubes are then removed simultaneously and immersed in a cold water bath for five minutes. The tubes are then removed from the water, dried and the contents of each tube are mixed by inverting them three or four times. The same colorimeter is employed for reading the absorbances of the fluids in tubes A, B and C, using the same filter for light having a wavelength of 540 millimicrons and maintaining the indicating device as reading zero absorbance for distilled water. The absorbances of tubes A, B and C are then read on the colorimeter and recorded. The concentration of globulin in the sample in grams of globulin per milliliters of sample is calculated by multiplying the difference between the absorbance of the sample (A) and that of the reagent blank (C) by 3.0 and dividing the product by the difference between the absorbance of the protein and globulin standard (B) and that of the reagent blank (C).

The serum samples of series II are analybed for total protein by the biuret reaction procedure of Henry et 211., Anal. Chem. 29, 1491 (1957) and for globulin concentration as determined by the salt fractionation procedure of Wolfson, Cohn et al., Am. J. Clin. Path., 18, 723 (1948). The total protein concentration, globulin concentration, and ratio of albumin to globulin results are compared with those obtained in series I.

Excellent agreement is observed between the results obtained in series I with the method and composition of the invention and the results obtained in series II by the art methods.

EXAMPLE 5 The reagent compositions employed above are held in a refrigerator at a temperature of about 3 C. for six months. At periodic intervals biuret protein reagent blanks are prepared, comprising the biuret reagent and sodium hydroxide of Example 4. The reagent blanks are heated and cooled as set out in Example 4 and then mixed with the globulin reagent of Example 3, to prepare a globulin reagent blank. Throughout the test period the absorbances of the biuret protein and globulin reagent blanks obtained from the aged reagents are not found to differ significantly from the absorbance originally observed with the freshly-prepared compositions.

EXAMPLE 6 In other determinations, the method and reagent of Example 4 are employed to compare samples of human serum containing known amounts of globulin and albumin with samples containing the same amount of globulin and albumin to which bilirubin has been added in known amounts. No significant difference in intensity of color is observed between the serum samples and the samples containing added bilirubin. The results of determination of total protein and globulin concentration are substantially the same for both sets of samples. These determinations indicate that no interference by bilirubin is observed at concentrations of 25 milligrams of bilirubin per 100 milliliters of sample.

EXAMPLE 7 In other determinations, the reagent and method of Example 4 are employed to compare the globulin concentration of samples containing known amounts of serum protein with identical samples containing the same amount of proteins to which known amounts of mixtures of albumin and globulin have been added. The results obtained are substantially identical with the concentration of globulin known to be present by addition of the known amounts to the samples.

EXAMPLE 8 The precision of the determination of total protein and of globulin from one determination to the other is evaluated with the method and reagents of Example 4. The evaluations are carried out by making determinations on samples containing different levels of total protein between and grams per 100 milliliters of sample, and levels of globulins between 2 and 12 grams per 100 milliliters of sample. In these operations, two consecutive determinations are carried out on forty pairs of identical samples. Statistical treatment of the data indicates excellent precision of the method from one determination to another.

EXAMPLE 9 The precision and repeatability of the determination of total protein and of globulin from one determination to the other is evaluated with the method and reagents of Example 4. The evaluations are carried out by making twenty-five consecutive determinations on identical samples containing different levels of total protein between about 6.3 and 8 grams per 100 milliliters of sample and levels of globulins between about 2.9 and 3.9 grams per 100 milliliters of sample. Statistical treatment of the data indicates excellent precision and repeatability of results from one determination to another.

What is claimed is:

1. A composition useful for the determination of globulin comprising a colorimetric amount of from about 5 to about percent (weight by volume) of paratoluenesulfonic acid and a sensitizing amount of cupric ion equivalent to from about 0.04 percent of cupric sulfate pentahydrate (weight by volume) to an amount of cupric ion suflicient to provide an optical density at 480600 millimicrons of about 0.2 therein, and a decolorizing amount of from about zero to about percent (by volume) of an analytically-acceptable strong acid dissolved in acetic acid.

2. The composition of claim 1 wherein the strong acid is sulfuric acid.

3. The composition of claim 1 wherein the cupric ion is present in an amount equivalent to from about 0.04 to about 0.2 percent by weight of cupric sulfate pentahydrate by volume of the mixture.

4. The composition of claim 3 wherein said analytically-acceptable strong acid is sulfuric acid and is present in a concentration of from about 5 to about 25 percent by volume.

5. A composition comprising a colorimetric amount of from about 5 to about 15 percent (weight by volume) of para-toluenesulfonic acid and a sensitizing amount of cupric ion equivalent to from about 0.04 to about 0.2 percent (weight by volume) of cupric sulfate pentahydrate, the paratoluene sulfonic acid and cupric ion being dissolved in acetic acid.

6. A composition of claim 5 further comprising from about 5 to about 25 percent by volume of sulfuric acid.

7. The composition of claim 6 wherein the composition contains from about 10 to about 20 percent by volume of sulfuric acid, from about to about percent by volume of acetic acid, from about 5 to about 15 percent (weight by volme) of para-toluene sulfonic acid and from about 0.04 to about 0.15 percent of cupric sulfate pentahydrate (weight by volume).

8. A method comprising:

(a) mixing a minor amount of a biological fluid containing an unknown amount of globulin with a colorimetric amount of para-toluenesulfonic acid and a sensitizing amount of cupric ion dissolved in acetic acid;

(b) heating the resulting mixture to a temperature of from about 90 to about C.; and

(c) cooling the heated mixture.

9. The method of claim 8 wherein the mixing step is carried out by mixing the biological fluid with a reagent composition comprising from about 10 to about 20 percent by volume of sulfuric acid, from about 80 to about 90 percent by volume of acetic acid, from about 5 to about 15 percent (weight by volume) of para-toluene sulfonic acid and an amount of cupric ion equivalent to from about 0.04 to about 0.15 percent of cupric sulfate pentahydrate (weight by volme).

10. The method of claim 8 wherein the biological fluid is a biological fluid which has been mixed with a biuret reagent, and further comprising the step of mixing the biological fluid with a decolorizing amount of an analytically-acceptable strong acid prior to the heating step.

11. The method of claim 10 wherein the ultimate mixture obtained after completion of the mixing steps comprises about one part by volume of the mixture of biological fluid and biuret reagent and from about three to about fifteen parts by volume of a mixture of the para-toluene sulfonic acid, cupric ion, acetic acid and analytically-acceptable strong acid.

References Cited Brackenridge, C. 1., Analytical Chemistry, vol. 32, pp. 1359-60 (1960).

Saifer, A., et al., Chemical Abstracts, vol. 56, p. 9037 (1962).

Welcher, F. J. ed., Standard Methods of Chemical Analysis, vol. II, Part A, pp. 11179 (1963).

MORRIS O. FOLK, Primary Examiner E. A. KATZ, Assistant Examiner US. Cl. X.R. 252408 

